An example of the use of fluorescence detection is in nucleic acid testing (NAT). This is a core element in molecular diagnostics for detecting genetic predispositions for diseases, for determining RNA expression levels or identification of pathogens, like bacteria and viruses that cause infections.
In many cases, particularly in the identification of pathogens, the amount of target DNA present in a reasonable sample volume is very low, and this does not allow direct detection. Amplification techniques are necessary to obtain detectable quantities of the target material. Different amplification techniques have been proposed and are used in daily practice. The most widely used are based on the so-called Polymerase chain reaction (PCR).
The amplification involves the denaturing of double-stranded DNA at elevated temperature (typically >90 degrees Celsius), specific binding of primers to the DNA sample at a reduced temperature (approximately 65 degrees) and copying of the original sequences starting from the primer position (at approximately 70 degrees). This procedure is repeated and in every cycle the amount of DNA with the specific sequence is doubled (when proceeding at 100% efficiency).
After amplification, the presence of target DNA is detected by measuring the fluorescence intensity of the labeled amplified DNA, for instance after electrophoretic separation in a capillary or after hybridization to so-called capture probes which are applied in spots on a surface over which the amplification product is flowed.
The standard technique for fluorescence detection is the use of a scanning confocal microscope. Typically, a small (<1 μm), diffraction limited spot is used to excite the fluorescence in the focal plane. In the detection part of the system, only the light resulting from this single excitation point is detected.
It has previously been proposed that the excitation of a number of spots or a complete line in parallel enables an increase in the scanning speed, without a major impact on the confocality of the detection system. A pixellated detector can be used to detect the fluorescent emission. However, it has also been suggested to use a more compact detector, based on the use of a simple photodiode in combination with a slit to allow confocal detection.
In order to generate the excitation beam for a confocal line scan, it has been proposed to modify an optical device for making a scan with a focused spot by adding an optical element such as a cylinder lens, that adds so-called astigmatism. If the cross-section of a beam is defined as the xy-plane, then each ray in the beam is characterized by coordinates (x,y). The beam is astigmatic if the rays on the x-axis, coordinates (x,0) have a different focus from the rays on the y-axis, coordinates (0,y).
With the use of cylinder lenses, light which is reflected from the sample and collected by the collection lens (objective lens) will no longer be a collimated beam. The light will always be divergent in at least one direction. This may require extra effort when the light is used for auto-focus or tracking purposes.
This divergence may also arises in wide field fluorescence microscopes. In such microscopes, the excitation light is defocused to illuminate a large area of the sample.